1. Technical Field
This invention pertains to the field of genetic manipulations of plants, and in particular to introduction of the aiiA gene into plants to quench pathogen quorum-sensing signaling. The invention also relates to transgenic plants expressing the aiiA gene with superior resistance to bacterial pathogens.
2. Description of the Background Art
Plant cells are held together by an intercellular substance (middle lamella) that is composed mainly of pectic compounds. Pathogenic bacterial cells enter plant tissues via wounds or stomata and are confined to the intercellular spaces of plant tissues in the early stage of infection. As infection progresses, pectolytic enzymes produced by the pathogen break down the connective tissue between cells and in cell walls, resulting in maceration symptoms in plants (also known as soft rot). See Collmer and Keen, 1986.
Bacterial plant pathogens produce an array of enzymes which attack host cell components, and these enzymes play important roles in suppression of host defense response and establishment of infection. In bacterial plant pathogens such as Erwinia carotovora, the OHHL quorum-sensing signal regulates the production of pectolytic enzymes, cellulase and proteases. See Pirhonen et al., 1993. Pectic polymers are structural constituents of plant primary cell walls and middle lamellae, providing physical support for turgid plant protoplasts, a barrier to bacterial penetration, and a source of host defense-eliciting oligalacturonates. See Collmer and Keen, 1986. Degradation of pectic polymers by the pectolytic enzymes has diverse effects on host-pathogen interactions.
Bacterial pathogens such as Erw. carotovora produce virulence factors, such as degradative enzymes, which assist the bacteria in entering plant cells and degrading plant tissues. The production of these factors is controlled by an elegant, density-dependent cell-cell communication mechanism known as quorum sensing. See Pirhonen et al., 1993. Several groups of signal molecules are involved in different microbial quorum sensing systems. See Fuqua et al., 1996; Robson et al., 1997. Among them, the best characterized are the N-acyl homoserine lactones (AHLs), also known as autoinducers (AIs). AHLs are members of a family of widely conserved signal molecules used in the quorum sensing systems of many Gram-negative bacteria. They also are involved in regulation of a diverse range of biological activities including expression of virulence genes of bacterial pathogens such as Erwinia carotovora, Erw. chrysanthemi, Erw. stewartii, Pseudomonas aeruginosa, and Xenorhabdus nematophilus. See Jones et al., 1993; Passador et al., 1993; Pirhonen et al., 1993; von Bodman and Farrand, 1995; Fuqua et al., 1996; Costa and Loper, 1997; Dunphy et al., 1997. AHLs act as ligands to a Lux-R family transcription factor. The LuxR-AHL-like complex controls the transcription of virulence genes. See Fuqua et al., 1996. AHL can couple to either a positive or a negative transcription factor for gene regulation. The list of pathogens using this signaling mechanism can be further expanded since there are many additional bacterial species that are known to produce AHLs. See Bassler et al., 1997; Cha et al., 1998; Dumenyo et al., 1998.
In the human pathogen P. aeroguinoisa, at least two different AI-signaling molecules, N-β-oxododecanoyl-L-homoserine lactone (ODHL) and N-butanoyl-L-homoserine lactone (BHL), and two transcriptional activators, LasR and RhlR/VsmR, influence the expression of a large number of virulence factors, including alkaline protease, elastase, exotoxin A, haemolysin and neuramimidase. See Pearson et al., 1994; Robson et al., 1997. The LasR-ODHL complex activates expression of both RhlR and the stationary-phase sigma factor RpoS in a regulatory cascade. See Latifi et al., 1995; Latifi et al., 1996; Pesci and Iglewski, 1997; McKnight et al., 2000; Whitely et al., 2000.
In plant pathogen P. stewartii, however, the negative transcription factor, EsaR, in conjunction with AHL, regulates virulence factor production. EsaR binds to its target promoters and inhibits transcription in the absence of N-βoxohexanoyl-L-homoserine lactone (OHHL). Binding of OHHL to EsaR releases its binding from the target promoter and thereby initiates the expression of the virulence factor. See von Bodman et al., 1998. In some other bacteria, such as Erw. carotovora, is not clear yet how AHL is involved in regulation of virulence factor production.
Although the target genes regulated by AHLs are extremely varied and regulatory mechanisms are likely diverse, the general mechanism of AHL-mediated quorum sensing signaling is very much conserved. The conserved feature of quorum sensing is that each individual cell produces a low level of quorum sensing molecules which are transported out of the cell. As the cell density increases and the accumulated signal reaches a threshold concentration (due to a high enough bacterial cell density) the AHLs are internalized and bind to their cognate LuxR-like transcription factors in the bacterial cells. This, in turn, activates the expression of virulence factor. Expression of virulence genes, therefore, is activated only when sufficient AHL signal molecules have accumulated. See Fuqua et al., 1996; Robson et al., 1997.
A newly discovered gene encoding a protein which inactivates AHL (aiiA) has been cloned from the Gram-positive bacterium Bacillus sp. strain 240B1. Expression of aiiA in Erw. carotovora strain SCG1, a pathogen which causes soft rot disease in many plants, significantly reduces the release of OHHL from the pathogen, decreases the extracellular pectrolytic enzyme activities, and attenuates pathogenicity in potato, eggplant, Chinese cabbage, carrot, celery, cauliflower, and tobacco (Dong et al., 2000). This confirms the important role of AHL in regulation of virulence genes.